Exercise is an ideal therapy for diabetes and obesity, but compliance is poor and how skeletal muscle contraction decreases metabolic disease risk is poorly understood. Abnormal lipid metabolism contributes to the pathophysiology of type 2 diabetes, but there is no consensus explanation for the relationship between lipids, muscle function, and metabolic decompensation. Unexpectedly, fatty acid synthase (FAS) is induced in skeletal muscle by high fat feeding and obesity in both animals and humans. Sarco/endoplasmic reticulum calcium ATPase (SERCA) is critical for normal muscle function. Skeletal muscle FAS deficiency causes high fat diet-induced muscle weakness because FAS is required to maintain SERCA activity by determining the phospholipid composition of the sarcoplasmic reticulum (SR). In young mice, a high fat diet is required to elicit weakness. The same phenotype due to the same mechanism occurs in aging mice with muscle FAS deficiency eating a low fat chow diet. FAS is linked to the phospholipid synthetic enzyme choline/ethanolamine phosphotransferase 1 (CEPT1). High fat feeding induces CEPT1 in skeletal muscle. Skeletal muscle CEPT1 deficiency causes high fat diet-induced muscle weakness through the same mechanism as FAS deficiency: altered SR phospholipid composition leading to decreased SERCA activity. FAS and CEPT1 in muscle appear to channel lipids predominantly to the SR since there is no effect on mitochondrial function, PPAR activation, ER stress or other processes in either FAS-deficient or CEPT1-deficient muscle. FAS is also linked to peroxisomal lipid synthesis. The final step in this process is mediated by Peroxisomal Reductase Activating PPAR (PexRAP), cloned and named based on its properties in nonmuscle tissue. PexRAP is a multifunctional enzyme capable of conventional phospholipid synthesis, and the phospholipid composition of muscle SR in PexRAP-deficient mice mirrors that of muscle SR in FAS and CEPT1 deficiency. In obese humans, FAS and CEPT1 are coordinately regulated. This pathway is dynamically modulated by weight loss, and related to insulin stimulated glucose disposal. Mass spectrometry analyses indicate that the SR phospholipid signature is similarly affected in muscle in FAS-deficient, CEPT1-deficient, and PexRAP- deficient mice, and in human metabolic syndrome. The long-term objective of this application is to characterize this novel link between diet, obesity, aging, and muscle function to improve the health of people with obesity and diabetes. We will test the hypothesis that an endogenous phospholipid synthetic pathway involving FAS, PexRAP, and CEPT1 in skeletal muscle channels lipids to maintain muscle function in the setting of metabolic stress. This hypothesis will be tested by addressing four aims: (1) To define the dynamics of lipogenic-mediated changes in skeletal muscle sarcoplasmic reticulum and calcium handling in response to changes in diet and exercise in mice. (2) To implicate FAS, PexRAP, and CEPT1 in a common phospholipid synthetic pathway leading to altered sarcoplasmic reticulum composition and function in cultured cells. (3) To determine if genetic inactivation of PexRAP in the skeletal muscle of mice alters the composition and function of the sarcoplasmic reticulum to affect strength and glucose metabolism. (4) To translate these observations to humans by determining if the composition and function of the sarcoplasmic reticulum is altered in people with the metabolic syndrome. Achieving the goals of this application could deliver new understanding of biochemical impediments to effective treatments, deliver novel biomarkers of progression to metabolic compromise in otherwise healthy obese people, and deliver viable targets for treating diabetes by repositioning drugs available through the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC).